Content of nitrate and nitrite in commercial and self‐made beetroot juices and the effect of storage temperature

Abstract Popularity of beetroot juice (BJ) is growing due to its high inorganic nitrate content NO3− and its potential physiological benefits. However, the content of NO3− is not indicated in most commercial BJs and it can be affected by seasonal changes and storage conditions. This study analyzed the content of NO3− and nitrite NO2− in five and two commercial and self‐made BJs, respectively, that were purchased in the summer and winter periods. The effect of storage temperature (20°C, 4°C, and −20°C) and pH was also analyzed. In nonconcentrated BJs, the NO3− content was 34 ± 20% (p = .075) in the winter than in the summer. NO3− was fully degraded in self‐made BJ after 3 days at 20°C. This effect was attenuated by 78% and 82% when it was kept at 4°C and −20°C, respectively. The addition of lemon juice (5%) to self‐made BJ was another useful approach to avoid NO3− degradation for 3 days when it was kept at 20°C. Regarding NO2−, self‐made BJ had higher concentration (0.097 ± 0.01 mg/mL) compared to commercial BJs (<0.1 mg/mL; p = .001). The pH of self‐made BJ was higher (6.3 ± 0.1) compared to commercial BJs (4.5 ± 0.3; p = .001). These results suggest that the content of NO3− in nonconcentrated BJs can substantially differ across the year and this is an important factor to take into account when recommending BJs to promote some of its potential physiological benefits.


| ME THODS
We analyzed the NO − 3 and NO − 2 content in five commercial BJs that are commonly used by professional and recreational athletes and two self-made BJs (Table 1).Commercial juices and raw beets were purchased in June 2021 and February 2022 and stored for less than 1 week at room temperature or under refrigeration as recommended by manufacturers before they were analyzed.

| Preparation of products
Self-made beetroot juice (SBJ) was prepared using whole beets (Beta vulgaris) from a local supermarket (Plymouth, UK).Beetroot was washed with tap water, and then with ultrapure water (Purelab OptionQ).The outer skin and inedible parts were removed before being chopped into small pieces and weighed using an electronic scale TA B L E 1 Beetroot juices analyzed in this study.(Precisa XB 3200C).Then, beetroot was juiced using an electric juicer machine (Waring 11JE65).Lemon was bought in a local supermarket and juiced using a fruit juicer.Then, 2.5 mL (5%) of lemon juice was mixed with 47.5 mL (95%) of fresh beetroot juice (SBJ), which is similar to the volume of lemon juice added into some commercial BJs analyzed in this study (Table 1).Commercial BJs were opened on the first day of analyses.All BJs were filtered using a Whatman® filter paper number

Brand
1 and centrifuged at 3500 rpm for 10 min to remove solid parts.

| Analysis of nitrate NO −
3 and nitrite NO − 2 All beetroot samples were centrifuged at 13,000 rpm at 4°C for 10 min before analysis.The content of NO − 3 and NO − 2 of each product was analyzed using a dedicated high-performance liquid chromatography analyzer (ENO-30; Eicom USA) as previously described (Corleto et al., 2018).Briefly, NO − 3 and NO − 2 were separated on a reverse-phase separation column packed with polystyrene polymer (NO-PAK 4.6 × 50 mm, EICOM; Amuza, Inc.), and NO − 3 was reduced to NO − 2 in a reduction column packed with copperplated cadmium filings (NO-RED EICOM; Amuza, Inc.).NO − 2 was mixed with a Griess reagent to form a purple azo dye in a reaction coil.The separation and reaction columns and the reaction coil were placed in a column oven set at 35°C.The absorbance of the color of the product dye at 540 nm was measured with a flow-through spectrophotometer (NOD-30; Eicom).The mobile phase (10% methanol, 0.15 M NaCl/NH 4 Cl, and 0.5 g/L 4Na-EDTA) and reactor phase (10% methanol, 1.25% HCl containing 5 g/L of sulfanilamide with 0.25 g/L of N-naphthylethylenediamine) were delivered at a flow rate of 0.33 mL/min and 0.10 mL/min, respectively.A standard curve was produced by injecting 10 μL of water with sodium NO − 3 (NaNO − 3 /7631-99-4; Sigma Aldrich) and sodium NO − 2 (NaNO − 2 /7632-00-0; Sigma Aldrich) at different concentrations (7.8, 15.6, 31.2, 62.5, 125, and 250 μM).Beetroot samples were diluted 1:200 using a carrier solution containing 10% methanol, 0.15 M NaCl/NH 4 Cl, and 0.5 g/L 4Na-EDTA.Samples were analyzed (10 μL) in duplicate on the first day and single on third and seventh day given the small coefficient of variation of NO − 3 (2.1 ± 1.9%) and NO − 2 (4.8 ± 3.0%) analyses.

| pH measurements
Measurements of pH were performed using a single-electrode digital pH meter (Lutron Electronic Enterprise Co Ltd.; Model PH-208) that was calibrated following the manufacturer's instructions prior to each use.

| Storage temperature
The effect of different storage temperatures on the NO − 3 and NO − 2 content was only analyzed in the first batch (June 2021).Eppendorf (1.5 mL) and Falcon tubes (3 mL) were filled with each product and kept at three different temperatures (20°C, 4°C, and −20°C) to analyze NO − 3 , NO − 2 , and pH on the first (baseline), third, and seventh day using the same methods described above.All the tubes were wrapped with aluminum foil to preserve the samples from light oxidation.Samples at −20°C were thawed on the same day of the analysis.Then, all samples were centrifuged at 13,000 rpm at 4°C for 10 min before analysis was undertaken.

| Statistical analyses
Data are presented as mean ± standard deviation.Differences in

NO −
3 and NO − 2 content and pH between different BJs were compared using a one-way analysis of variance.Post hoc analyses were performed using Tukey HSD.Data were analyzed using the statistical software SPSS (version 28).The level of significance was set at p < .05.

Using the NO −
3 results from each product, we calculated the amount of BJ that was needed to achieve the minimum dose of NO − 3 to enhance exercise performance (5 mmol of NO − 3 = 310 mg) (Figure 2).With the exception of concentrated (JW2) and self-made juice (SBJ), an average of 258 ± 162 mL more BJ from the winter batches of commercial BJs was needed to achieve such amount compared to the summer batches.

, nitrite, and pH
The content of NO − 3 in juices stored at 20, 4, and −20°C for 1, 3, and 7 days during the summer is shown in Figure 3.
A reduction of 24% (from 6.3 ± 0.2 to 4.8 ± 0.2 mg/mL; p < .001)and 46% (from 6.3 ± 0.2 to 3.4 ± 0.2 mg/mL, p < .001) in NO − 3 was observed when concentrated BJ (JW2) was kept at 20°C for 3 and 7 days, respectively (Figure 3A).A similar effect was observed when JW2 was kept for 3 days at 4°C (from 6.3 ± 0.2 to 4.7 ± 0.2 mg/mL; because wet conditions (rainfalls) can wash out NO − 3 into the groundwater, a phenomenon known as NO − 3 leaching (Thomas et al., 2019).For this reason, it is feasible to use additional fertilizer (nitrogen) in autumn and winter in some vulnerable areas to improve the crops yield (Thomas et al., 2019).Four of the commercial BJs (JW1, JW2, BN, and BT) analyzed in this study indicated that beetroot used was organic so nitrogen fertilizers were not supposed to be used during the growth of the crops.Interestingly, all of them, except the concentrated juice (JW2), had lower content of NO − 3 when they were bought and analyzed in the winter, which may suggest that beetroot were grown over the summer.However, this information was not provided by the commercial companies.Light conditions, use of organic matter (animal manure), and storage conditions are also important factors to take into account as they can affect the content of NO − 3 in vegetables (Dechorgnat et al., 2010;Gov.UK, 2022;Santamaria, 2006).Commercial companies can obtain beetroot from different locations and areas given the large amount of product needed to constantly supply the market, which can modify the content of NO − 3 in the final product.Furthermore, there is no regulation about labeling the content of NO − 3 in commercial BJ, its origin, or when crops were harvested.This is relevant given the potential physiological implications of NO − 3 and the variations in the content of this ion observed in this study in some commercial products.
Although individuals can always choose to consume larger-thanrecommended amounts, potential disadvantages to doing so include increased cost, greater volume to ingest, higher intake of oxalate, and potential side effects.
Only two of the commercial juices analyzed in this study (JW1 and JW2) reported an estimated value of NO − 3 in the serving size (Table 1).The first juice (JW1) claimed that the NO − 3 content was on average 800 mg per liter (0.8 mg/mL).Compared to this, we found that the NO − 3 content of this product was 38% higher in the summer batch (June 2020) (1.1 mg/mL) and 14% lower in the winter batch (February 2021) (0.69 mg/mL).On the other hand, the NO − 3 content from a concentrated product from the same commercial brand (JW2) was 47% and 50% higher in summer and winter batches compared to the claimed NO − 3 content.Our results are in agreement with a previous study indicating that the NO − 3 content of the same BJ was 23% higher than the claimed NO − 3 content (Weightman et al., 2012).However, they did not compare the content of NO − 3 of the same product across different periods of the year.Furthermore, both studies showed that commercial concentrated beetroot shots (JW2) had nearly five times more NO − 3 than commercial nonconcentrated and fresh BJ.Concentrated beetroot shots appeared in the market a decade ago to provide the minimal dose of inorganic NO −

3
(5 mmol = 310 mg/serving) that has been suggested to enhance exercise capacity in a small volume (Santamaria et al., 1999).Although the method to concentrate BJ is not reported on the label, this process is usually performed by removing part of the water from the juice (Jones et al., 2018).
Regarding the effect of temperature storage on the content of NO − 3 and NO − 2 , rapid degradation of NO − 3 occurred in SBJ when it was kept at 20°C for 3 days, but this reaction was attenuated by storing it at low temperatures and by adding lemon juice (SBJL), a natural source of ascorbic acid.This is in agreement with our hypothesis, suggesting that low temperatures and the addition of lemon juice can help to attenuate NO − 3 degradation in BJ.Ascorbic acid is widely used in the food industry for its antioxidant and stabilizing properties (Bazaria & Kumar, 2016).Two of the commercial juices analyzed in this study (JW2 and BN) contained lemon juice, two more apple juice (JW1 and CW), and another one (CW) was fortified with ascorbic acid.Despite the addition of lemon juice into concentrated BJ (JW2), we found a rapid reduction in the content of NO − 3 occurred over days 3 and 7 that was not attenuated at low temperatures.
According to this, rapid consumption of concentrated BJ is recommended to enhance NO − 3 intake.This is in agreement with the recommendations from the commercial companies indicating to keep the juice refrigerated and consume it within 3-7 days once opened.
The addition of lemon and apple juices can also help to enhance the organoleptic characteristics of BJ for some people who dislike the taste of beetroot (Grand View Research, 2022).
The content of NO − 2 was very low (<0.1 mg/mL) in all the juices at baseline; however, a rapid increase was observed in SBJ on day 3 at 20°C.This could happen due to the activity of NO − 3 reductase enzymes or microorganisms present in beetroot as the decrease of NO − 3 was accompanied by the increase of NO − 2 .From a safety point of view, the levels of NO − 2 achieved on day 3 were quite low to cause harm in healthy individuals as doses above 100 mg/kg of body mass are required to produce serious side effects in humans (Liao & Seib, 1988).According to our results, the consumption of over 4 L of BJ rich in NO − 2 over a relatively short period of time may be needed to reach this quantity of NO − 2 .However, a word of caution is needed about BJ overload among athletes thinking 'the more the better'.
The pH of commercial BJs was more acidic than self-made juice (SBJ), which can be related to lacto-fermentation and addition of ascorbic acid in commercial juices.Two commercial juices of this study were lacto-fermented (BN and BT), which consists of the addition of lactic acid bacteria consuming sugars to produce acid compounds and carbon dioxide by fermentation (Klewicka et al., 2015).
Three commercial juices also contained lemon juice (JW2 and BN) or vitamin C as an additive (CW).The addition of lemon juice to SBJ (SBJL) is a useful approach to maintain the content of NO − 3 as we demonstrated in this study.On the other hand, further research is needed to investigate whether lacto-fermentation and/or addition of other juices can modify NO − 3 bioavailability.This study had some limitations that are worth discussing.
First, it was based on a Master's thesis that was performed during Covid-19 pandemic when students had to deal with laboratory restrictions.Bottles of five of the most consumed brands of BJ in the UK were analyzed in the summer (June) and winter (February) seasons.The batch code of each product was not recorded, but we believe that juices belonged to different batches given the time gap (8 months) between the purchase and analyses of them.
Despite this limitation, our results are still interesting, indicating that the content of NO − 3 can substantially differ especially in commercial nonconcentrated BJs.We also had limitations to increase

F
I G U R E 2 Estimated amount of beetroot juice required to achieve 5 mmol of nitrate NO − 3 from commercial and self-made beetroot juices in the summer and winter.F I G U R E 1 Content of nitrate NO − 3 (A), nitrite NO − 2 (B), and pH (C) in commercial and self-made beetroot juices in two different periods of the year.(a represents statistical differences between beetroot juices; b represents statistical differences between beetroot juice batches in the summer and winter.